Role of Arf GTPases in pathogenesis of citomegalovirus infection (Arf & CMV) 

Grantor: Croatian Science Foundation 
Project duration: 2021. do 2025. 
Total funding:  679.000,00 HRK 

Project team 

Principal investigator  

• prof. dr. sc. Gordana Blagojević Zagorac, dr. med. 

Research team: 

• izv. prof. dr. sc. Tamara Gulić mag. biol.  
• Valentino Pavišić dr. med.  
• Silvija Lukanović Jurić dr.med. 
• Natalia Jug Vučko, mag. pharm. inv. 

Brief description  

Arf proteins are small GTPases that, through the recruitment of effector proteins, organize the cell cytoskeleton and change the dynamics of membranes, thus regulating transport within the secretory and endocytic systems, cell division and migration. Arf proteins are divided into three classes based on their sequence homology. Class I includes Arf1, Arf2 and Arf3, class II Arf4 and Arf5, while Arf6 is the only representative of class III. Just like other small GTPases, Arf proteins function as molecular switches because they cycle between their inactive (GDP-bound) and active (GTP-bound) forms. When inactivated, Arfs are mainly diffused throughout the cytoplasm, and are activated on cell membranes at sites where guanine nucleotide exchange factors (GEFs) are found. GEF proteins mediate the exchange of GDP for GTP, which causes conformational changes of Arf proteins and results in their tight binding to membranes and the recruitment of effector molecules located in close proximity. The endogenous GTPase activity of Arf proteins is extremely low and therefore GAP proteins are required for their inactivation, which, by stimulating the hydrolysis of GTP, lead to their inactivation. After hydrolysis of GTP, Arf1 and Arf3 are released from the membranes, while Arf4, Arf5, and Arf6 may in some cases still remain bound to the membrane. 

Cytomegaloviruses (CMV) are widespread DNA viruses from the herpesvirus family that, after acute, mostly asymptomatic infection, enter a latent phase from which they can be reactivated at any time and lead to life-threatening conditions in immunocompromised people. Already during the early phase of CMV infection, there is a massive reorganization of cellular functions, including the reorganization of membrane compartments and the cell cytoskeleton, which ultimately results in the formation of a cytoplasmic virion assembly compartment (cVACa). cVAC is a large, cylindrical, juxtanuclear-located compartment that has phenotypic characteristics of many intracellular compartments, including early endosomes, recycling endosomes, and the trans-Golgi network (TGN). Although the function of Arf proteins has been investigated in the pathogenesis of many bacterial and viral infections, and it is known that the activation of Arf1 and Arf6 proteins is an important factor in the virulence of L. pneumophila, R. prowazekii and  Salmonella bacteria  and single-stranded RNA viruses such as Hepatitis C virus, coronavirus and HIV, very little is known about the role of Arf protein in CMV infection. Unlike other pathogens that only use the cellular machinery to run their own life cycle, the characteristic of herpesviruses is extensive rearrangement of host cell organelles and in some cases even the formation of new compartments, which is the characteristic of CMV. Therefore, it is logical to expect that Arf proteins will be an important factor in the virulence of cytomegaloviruses, i.e. the entire herpesvirus family. 

This research would greatly contribute to the understanding of the role of Arf proteins in the replication cycle of CMV, that is, this research would determine whether Arf proteins are necessary for the entry of the virus into the cell, its replication and the formation of cVAC. Furthermore, through the analysis of the most important Arf GEF and Arf GAP proteins that interact with Arf proteins during CMV infection, and which are responsible for their activation/inactivation, new knowledge about the specificity and localization of Arf-regulatory proteins would be analysed, which could also apply to uninfected cells. Furthermore, a major breakthrough for this research would be the determination of the gene region of the virus, responsible for the reorganization of the Arf system of host cells during CMV infection. Understanding how CMVs lead to reorganization of the endosomal system of the host cell, which results in evasion of immune response and viral replication, would create a platform for development of new antiviral drugs. 

Publications

1. Pavišić V, Mahmutefendić Lučin H, Blagojević Zagorac G, Lučin P. Arf GTPases Are Required for the Establishment of the Pre-Assembly Compartment in the Early Phase of Cytomegalovirus Infection. Life (Basel). 2021 Aug 23;11(8):867. doi: 10.3390/life11080867. PMID: 34440611; PMCID: PMC8399710.
2. Mahmutefendić Lučin H, Blagojević Zagorac G, Marcelić M, Lučin P. Host Cell Signatures of the Envelopment Site within Beta-Herpes Virions. International Journal of Molecular Sciences. 2022; 23(17):9994. https://doi.org/10.3390/ijms23179994
3. Lučin P, Mahmutefendić Lučin H, Blagojević Zagorac G. Cytomegaloviruses reorganize endomembrane system to intersect endosomal and amphisome-like egress pathway. Front Cell Dev Biol. 2023 Dec 19;11:1328751. doi: 10.3389/fcell.2023.1328751. PMID: 38178873; PMCID: PMC10766366.

Proceedings Paper

1. Valentino Pavišić, Natalia Jug Vučko, Hana Mahmutefendić Lučin, Ljerka Karleuša, Pero Lučin, Gordana Blagojević Zagorac. Cytomegaloviruses induce massive reorganization of Arf proteins in theearly phase of infection. Abstracts of ECI 2021; 6th European Congress of Immunology. 1‐4 September 2021. Virtual meeting. (European Journal of Immunology Volume 51, Suppl. 1, August 2021)
2. Valentino Pavišić, Hana Mahmutefendić Lučin, Tamara Gulić, Natalia Jug Vučko, Pero Lučin1,2 and Gordana Blagojević Zagorac. MCMV induces activation and accumulation of Arf6 GTPase on membranes of virion assembly compartment. 2021 Annual Meeting of the Croatian Immunological Society, Trogir, 23.-25.09.2021.
3. Valentino Pavišić, Hana Mahmutefendić Lučin, Natalia Jug Vučko, Pero Lučin, Gordana Blagojević Zagorac. Arf proteins and their GEFs are generally excluded from outer part of murine cytomegalovirus assembly compartment in the late phase of MCMV infection15^thAnnual Meeting of Croatian Physiological Society with lnternational Participation, Zagreb 07-08.10.2021.
4. Valentino Pavišić, Hana Mahmutefendić Lučin, Pero Lučin, Gordana Blagojević Zagorac. Arf proteins accumulate in the citomegalovirus assembly compartment during the early phase of infection. The 45th FEBS Virtual Congress. 3-8 July 2021. Virtual meeting.
5. Pavišić, Valentino; Mahmutefendić Lučin, Hana; Lučin, Pero; Blagojević Zagorac, Gordana. Arf1 and Arf6 Are Required for the Establishment of Murine Cytomegalovirus Infection. 46^th International Herpesvirus Workshop. Virtual meeting, SAD, 25.7. – 29.7. 2022.
6. Pavišić, Valentino; Mahmutefendić Lučin, Hana; Lukanović Jurić, Silvija; Lučin, Pero; Blagojević Zagorac, Gordana. Arf1 and Arf6 GTPases are required for intracellular transport of cytomegalovirus virions. 3^rd Regional Congress of Physiological Societies and 5^th Congress of Croatian Physiological Society. Plitvice, Hrvatska, 22.09. – 25.09. 2022.
7. Blagojević Zagorac, Gordana; Karleuša, Ljerka; Viduka, Ivona; Jug Vučko, Natalia; Pavišić, Valentino; Mahmutefendić Lučin, Hana; Lučin, Pero. BIG, GBF and BRAG ARF GEFS are required for the progression of immediate-early and early phase of MCMV infection. Congress of the Croatian Society of Biochemistry and Molecular Biology HDBMB22: From Science to Knowledge. Brela, Hrvatska, 28.09. – 01.10. 2022.
8. Blagojević Zagorac, Gordana; Gulić, Tamara; Pavišić, Valentino; Mahmutefendić Lučin, Hana; Lukanović Jurić, Silvija; Lučin, Pero. Activation of Arf GTPases during murine cytomegalovirus infection. Annual meeting of the Croatian Immunological Society 2022. Sveti Martin na Muri, Hrvatska, 06.10. – 08.10. 2022.
9. Štimac, Igor; Blagojević Zagorac, Gordana; Marcelić, Marina; Lučin, Pero; Mahmutefendić Lučin, Hana. Rab10 GTPase is involved in membrane remodeling to form assembly compartment in the early phase of murine cytomegalovirus infection. 3^rd Regional Congress of Physiological Societies and 5^th Congress of Croatian Physiological Society. Plitvice, Hrvatska, 22.09. – 25.09. 2022.
10. Viduka, Ivona; Štimac, Igor; Gulić, Tamara; Blagojević Zagorac, Gordana; Mahmutefendić Lučin, Hana; Lučin, Pero. Suppression of Early Endosomal Sorting Nexins Affects Murine Cytomegalovirus Envelopment and Egress. 46^th International Herpesvirus Workshop. Virtual meeting, SAD, 25.7. – 29.7. 2022.
11. Marcelić, Marina; Mahmutefendić Lučin, Hana; Jurak Begonja, Antonija; Blagojević Zagorac, Gordana; Viduka, Ivona; Lučin, Pero. Endosomal Phosphatidylinositol-3-phosphate is Essential for the Final Assembly of CMV Virions. 46^th International Herpesvirus Workshop. Virtual meeting, SAD, 25.7. – 29.7. 2022.
12. Štimac, Igor; Radić, Barbara; Blagojević Zagorac, Gordana; Marcelić, Marina; Lisnić, Berislav; Lučin, Pero; Mahmutefendić Lučin, Hana. EHBP1 and Rabin8 GTPase recruit Rab10 GTPase in Murine cytomegalovirus assembly compartment. The Annual Meeting of the Croatian Immunological Society. , Hrvatska, 05.10. – 07.10. 2023.